Bovine kidney pyruvate dehydrogenase complex. Limited proteolysis and molecular structure of the lipoate acetyltransferase component.
1. Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated by elastase in a similar manner as described earlier for papain. The core component, lipoate acetyltransferase, is cleaved by elastase into an active fragment (Mr 26000) and a fragment with apparent Mr of 45000 as analyzed by dodecylsulfate gel electrophoresis. Due to the fragmentation of the core, the enzyme complex is disassembled into its component enzymes which retain their complete enzymatic activities as assayed separately. 2. A different mechanism was found for the inactivation of pyruvate dehydrogenase complex with trypsin and some other proteases (chymotrypsin, clostripain). In these cases, the pyruvate dehydrogenase component is inactivated rapidly by limited proteolysis. More slowly, the enzyme complex is disassembled simultaneously with fragmentation of the lipoate acetyltransferase which again results in an active fragment of Mr 26000 and another fragment of apparent Mr 45000. Upon prolonged proteolysis, the latter fragment is cleaved further to give products of Mr 36000 or lower. 3. The enzyme-bound lipoyl residues of the pyruvate dehydrogenase complex have been labelled covalently by incubation with [2-14C]pyruvate. After treatment of this [14C]acetyl-enzyme with papain, elastase, or trypsin, radioactivity was associated exclusively with the 45000-Mr and 36000-Mr fragments but not with the active 26000-Mr fragment. 4. It is concluded that the bovine kidney lipoate acetyltransferase core is composed of 60 subunits each consisting of two dissimilar folding domains. One of these contains the intersubunit binding sites as well as the active center for transacylation whereas the other possesses the enzyme-bound lipoyl residues.
D000123:Acetyltransferases; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D010450:Endopeptidases; D007668:Kidney / Q000201:enzymology*; D008970:Molecular Weight; D010196:Pancreatic Elastase; D010206:Papain; D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid / Q000031:analogs & derivatives; D014357:Trypsin
D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D010450:Endopeptidases; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D003546:Cysteine Endopeptidases; D010206:Papain; C019913:clostripain